Binding of three ruthenium(II) compounds of general formula mer-[Ru(L3)(N-N)X][Y] (where L3 = 4′-chloro-2,2′:6′,2′′-terpyridine (Cl-tpy); N-N = 1,2-diaminoethane (en), 1,2-diaminocyclohexane (dach) or 2,2′-bipyridine (bipy); X = Cl; Y = Cl) to human serum albumin (HSA) has been investigated by nano-LC/nano-ESI MS and docking studies. A bottom-up proteomics approach has been applied for the structural characterization of metallated proteins and the data were analyzed in both the positive and negative ion mode. The negative ion mode was achieved after the post-column addition of an isopropanol solution of formaldehyde that enabled sample ionization at micro-flow rates. The negative ion mode MS has been proved to be beneficial for the analysis of binding sites on ruthenated protein in terms of ion charge reduction and consequent simplification of target sequence identification based on isotopic differences between ruthenated and non-ruthenated peptides. Moreover, the negative ion mode ESI MS shows the advantage of singly charged ion formation and, unlike MALDI MS, it does not cause complete ligand fragmentation, merging the benefits of each method into a single experiment. Six target sequences were identified for the binding of en and dach compounds, and four sequences for the binding of bipy. All compounds have been found to bind histidine and one aspartate residue. Docking studies showed that the identified sequences are the constituents of five distinct binding sites for en and dach, or two sites for the bipy complex. The selection of binding sites seems to be dependent on the chelate ligand and the form of the complex prior or after hydrolysis of the leaving chloride ligand.

中文翻译：

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对接研究支持的钌化血清白蛋白的正负纳米电喷雾质谱：定义蛋白质上金属药物结合位点的综合方法†

三种通式为mer- [Ru（L3）（NN）X] [Y]的钌（II）化合物的结合（其中L3 = 4'-chloro-2,2'：6'，2''-叔吡啶（Cl -tpy）; NN＝ 1,2-二氨基乙烷（en），1,2-二氨基环己烷（dach）或2,2'-联吡啶（联吡啶）；X = Cl; Y = Cl）与人血清白蛋白（HSA）的关系已通过nano-LC / nano-ESI MS和对接研究进行了研究。自下而上的蛋白质组学方法已应用于金属化蛋白质的结构表征，并且在正离子和负离子模式下都对数据进行了分析。柱后添加甲醛的异丙醇溶液可实现负离子模式，该溶液可使样品以微流速电离。负离子模式MS已被证明有利于分析钌蛋白上的结合位点，从而减少离子电荷，从而简化了基于钌和非钌肽之间的同位素差异的靶序列鉴定。而且，负离子模式ESI MS显示出形成单电荷离子的优势，并且与MALDI MS不同，它不会引起配体完全断裂，将每种方法的优点合并到一个实验中。鉴定了六个结合en和dach化合物的靶序列，和四个结合了bipy的序列。已经发现所有化合物都结合组氨酸和一个天冬氨酸残基。对接研究表明，鉴定出的序列是en和dach的五个不同结合位点的组成，或者是bipy复合物的两个位点的组成。结合位点的选择似乎取决于螯合配体和在离开的氯化物配体水解之前或之后络合物的形式。将每种方法的优点合并到一个实验中。鉴定了六个结合en和dach化合物的靶序列，和四个结合了bipy的序列。已经发现所有化合物都结合组氨酸和一个天冬氨酸残基。对接研究表明，鉴定出的序列是en和dach的五个不同结合位点的组成，或者是bipy复合物的两个位点的组成。结合位点的选择似乎取决于螯合配体和在离开的氯化物配体水解之前或之后络合物的形式。将每种方法的优点合并到一个实验中。鉴定了六个结合en和dach化合物的靶序列，和四个结合了bipy的序列。已经发现所有化合物都结合组氨酸和一个天冬氨酸残基。对接研究表明，鉴定出的序列是en和dach的五个不同结合位点的组成，或者是bipy复合物的两个位点的组成。结合位点的选择似乎取决于螯合配体和在离开的氯化物配体水解之前或之后络合物的形式。对接研究表明，鉴定出的序列是en和dach的五个不同结合位点的组成，或者是bipy复合物的两个位点的组成。结合位点的选择似乎取决于螯合配体和在离开的氯化物配体水解之前或之后络合物的形式。对接研究表明，鉴定出的序列是en和dach的五个不同结合位点的组成，或者是bipy复合物的两个位点的组成。结合位点的选择似乎取决于螯合配体和在离开的氯化物配体水解之前或之后络合物的形式。